In addition to their known uses in diagnostics, antibodies have been shown to be useful as therapeutic agents. For example, immunotherapy, or the use of antibodies for therapeutic purposes has been used in recent years to treat cancer. Passive immunotherapy involves the use of monoclonal antibodies in cancer treatments. See for example, Cancer: Principles and Practice of Oncology, 6th Edition (2001) Chapt. 20 pp. 495-508. These antibodies can have inherent therapeutic biological activity both by direct inhibition of tumor cell growth or survival and by their ability to recruit the natural cell killing activity of the body's immune system. These agents can be administered alone or in conjunction with radiation or chemotherapeutic agents. Rituximab and Trastuzumab, approved for treatment of non-Hodgkin's lymphoma and breast cancer, respectively, are two examples of such therapeutics. Alternatively, antibodies can be used to make antibody conjugates where the antibody is linked to a toxic agent and directs that agent to the tumor by specifically binding to the tumor. Gemtuzumab ozogamicin is an example of an approved antibody conjugate used for the treatment of leukemia. Monoclonal antibodies that bind to cancer cells and have potential uses for diagnosis and therapy have been disclosed in publications. See, for example, the following patent applications which disclose, inter alia, some molecular weights of target proteins: U.S. Pat. No. 6,054,561 (200 kD c-erbB-2 (Her2), and other unknown antigens 40-200 KD in size) and U.S. Pat. No. 5,656,444 (50 kD and 55 kD oncofetal protein). Example of antibodies in clinical trials and/or approved for treatment of solid tumors include: Trastuzumab (antigen: 180 kD, HER2/neu), Edrecolomab (antigen: 40-50 kD, Ep-CAM), Anti-human milk fat globules (HMFG1) (antigen>200 kD, HMW Mucin), Cetuximab (antigens: 150 kD and 170 kD, EGF receptor), Alemtuzumab (antigen: 21-28 kD, CD52), and Rituximab (antigen: 35 kD, CD20).
The antigen targets of trastuzumab (Her-2 receptor), which is used to treat breast cancer, and cetuximab (EGF receptor), which is in clinical trials for the treatment of several cancers, are present at some detectable level on a large number of normal human adult tissues including skin, colon, lung, ovary, liver, and pancreas. The margin of safety in using these therapeutics is possibly provided by the difference in the level of expression or in access of or activity of the antibody at these sites.
Another type of immunotherapy is active immunotherapy, or vaccination, with an antigen present on a specific cancer(s) or a DNA construct that directs the expression of the antigen, which then evokes the immune response in the individual, i.e., to induce the individual to actively produce antibodies against their own cancer. Active immunization has not been used as often as passive immunotherapy or immunotoxins.
Several models of disease (including cancer) progression have been suggested. Theories range from causation by a single infective/transforming event to the evolution of an increasingly “disease-like” or ‘cancer-like’ tissue type leading ultimately to one with fully pathogenic or malignant capability. Some argue that with cancer, for example, a single mutational event is sufficient to cause malignancy, while others argue that subsequent alterations are also necessary. Some others have suggested that increasing mutational load and tumor grade are necessary for both initiation as well as progression of neoplasia via a continuum of mutation-selection events at the cellular level. Some cancer targets are found only in tumor tissues, while others are present in normal tissues and are up regulated and/or over-expressed in tumor tissues. In such situations, some researchers have suggested that the over-expression is linked to the acquisition of malignancy, while others suggest that the over-expression is merely a marker of a trend along a path to an increasing disease state.
An ideal diagnostic and/or therapeutic antibody would be specific for an antigen present on a large number of cancers, but absent or present only at low levels on any normal tissue. The discovery, characterization, and isolation of a novel antigen that is specifically associated with cancer(s) would be useful in many ways. First, the antigen could be used to make monoclonal antibodies against the antigen. An antibody would ideally have biological activity against cancer cells and be able to recruit the immune system's response to foreign antigens. An antibody could be administered as a therapeutic alone or in combination with current treatments or used to prepare immunoconjugates linked to toxic agents. An antibody with the same specificity but with low or no biological activity when administered alone could also be useful in that an antibody could be used to prepare an immunoconjugate with a radio-isotope, a toxin, or a chemotherapeutic agent or liposome containing a chemotherapeutic agent, with the conjugated form being biologically active by virtue of the antibody directing the toxin to the antigen-containing cells.
One aspect desirable for an ideal diagnostic and/or therapeutic antibody is the discovery and characterization of an antigen that is associated with a variety of cancers. There are few antigens that are expressed on a number of types of cancer (e.g., “pan-cancer” antigen) that have limited expression on non-cancerous cells. The isolation and purification of such an antigen would be useful for making antibodies (e.g., diagnostic or therapeutic) targeting the antigen. An antibody binding to the “pan-cancer” antigen could be able to target a variety of cancers found in different tissues in contrast to an antibody against an antigen associated with only one specific type of cancer. The antigen would also be useful for drug discovery (e.g., small molecules) and for further characterization of cellular regulation, growth, and differentiation.
Transferrin receptor is broadly expressed in human tumors (Gatter et al., Transferrin receptors in human tissues: their distribution and possible clinical relevance, J Clin Pathol 36, 539-545 (1983)) and plays a key role in cell proliferation and survival. Antibodies that bind to the transferrin receptor have previously been shown to be efficacious in animal tumor models. In a leukemia xenograft model using CCRF-CEM cells (White et al., Combinations of anti-transferrin receptor monoclonal antibodies inhibit human tumor cell growth in vitro and in vivo: evidence for synergistic antiproliferative effects, Cancer Res 50, 6295-6301 (1990)) and in a M21 human melanoma xenograft (Trowbridge & Domingo, Anti-transferrin receptor monoclonal antibody and toxin-antibody conjugates affect growth of human tumor cells, Nature 294, 171-173 (1981)), transferrin receptor antibodies also inhibited tumor progression.
The transferrin receptor has been studied as a cancer target since the 1980s using naked antibodies, toxin conjugated antibodies and transferrin-toxin conjugates (see, e.g., Griffin et al., Combined antitumor therapy with the chemotherapeutic drug doxorubicin and an anti-transferrin receptor immunotoxin: In vitro and in vivo studies, J Immunol 11, 12-18 (1992); Qian et al., Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway, Pharmacological Reviews 54, 561-587 (2002); Trowbridge & Collin et al., Structure-function analysis of the human transferrin receptor: Effects of anti-receptor monoclonal antibodies on tumor growth, Curr Stud Hematol Blood Transf 58, 139-147 (1991)), including a phase I clinical trial with the murine IgA antibody 42/6 (Brooks et al., Phase Ia trial of murine immunoglobulin A antitransferrin receptor antibody 42/6, Clin Cancer Res 1, 1259-1265 (1995)). Expression of the transferrin receptor is correlated with cell proliferation and it has been suggested that this accounts for the high proportion of tumors that stain positively with transferrin receptor antibodies and limited staining of normal tissues (Gatter, 1983). It is generally accepted that transferrin receptor antibodies inhibit cell proliferation by reducing the uptake of iron into the cell (Kemp, Iron deprivation and cancer: a view beginning with studies of monoclonal antibodies against the transferrin receptor, Histol Histopathol 12, 291-296, (1997)). This can be achieved by blocking the interaction of the transferrin receptor with iron-charged transferrin or by altering the dynamics of transferrin receptor cycling and cell surface presentation. The effect of blocking iron uptake in tumor cells manifests initially as a cell cycle arrest, primarily in S-phase followed by an accumulation of G1 phase cells (White, 1990). The ultimate endpoint of iron withdrawal appears to vary from cytostasis to the induction of cell death.
Rat derived antibody that recognizes the murine transferrin receptor was tested in a syngeneic mouse leukemia model (Savage, et al., Effects of monoclonal antibodies that block transferrin receptor function on the in vivo growth of a syngeneic murine leukemia, Cancer Res 47, 747-753 (1987)). This molecule significantly improved survival relative to controls and there was no evidence of gross toxicity or evidence of damage to normal tissues recognized by the antibody over a four week treatment period. Additionally, there were no changes in erythrocyte or white blood cell counts. However, an analysis of bone marrow progenitor cells showed a two fold decrease in CFU-e/106 cells and a less pronounced reduction in CFU-c. Additional insight into the effect(s) of blocking the transferrin receptor can be provided by evaluating the results of a phase I clinical trial that was performed using the mouse antibody 42/6. In this study, there was evidence of mixed tumor responses, despite the short treatment course and poor pharmacokinetics of the mouse antibody (Brooks, 1995). An evaluation of patients treated with 42/6 showed evidence of reduced marrow BFU-e after treatment with the antibody, but the result was not statistically significant. Because transferrin receptor has been shown to be expressed on differentiating bone marrow progenitor cells (Helm et al., Characterization and phenotypic analysis of differentiating CD34+ human bone marrow cells in liquid culture, Eur J Haematol 59, 318-326 (1997)) it is desirable that a therapeutic agent incorporating an anti-transferrin receptor antibody have a potential therapeutic effect that outweighs the potential for bone marrow toxicity.
What is needed are novel targets on the surface of diseased and/or cancer cells that may be used to diagnose and treat such diseases and/or cancers with antibodies and other agents which specifically recognize the cell surface targets. There exists a further need, based on the discoveries disclosed herein, for novel antibodies and other agents which specifically recognize targets on the surface of cells that can modulate, either by reducing or enhancing, the disease-promoting activities of transferrin receptor. It is an object of this invention to identify antagonists of human transferrin receptor that are capable of inhibiting its disease-associated activities. It is another object to provide novel compounds for use in the assay of transferrin receptor, and for use as immunogens or for selecting anti-human transferrin receptor antibodies.
As will be described in more detail below, the present inventors have discovered a novel epitope of the human transferrin receptor, identified as the antigen target of the novel antagonists, modulators and antibodies provided herein.